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12.4: Microtubules - Biologi

12.4: Microtubules - Biologi


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Mikrotubul terdiri daripada dua subunit globular yang sama rata, struktur dan serupa: tubulin α dan β. Seperti mikrofilamen, mikrotubulus juga bergantung pada nukleotida trifosfat untuk pempolimeran, tetapi dalam kes ini, itu adalah GTP.

Kestabilan mikrotubulus bergantung pada suhu: jika disejukkan hingga 4 ° C, mikrotubulus jatuh menjadi heterodimer αβ-tubulin. Dihangatkan semula hingga 37 ° C, tubulin repolimerisasi jika terdapat GTP.

Kesamaan lain ialah mikrotubulus mempunyai kekutuban di mana hujung (-) jauh lebih aktif daripada hujung (+). Walau bagaimanapun, tidak seperti mikrofilamen pasangan berpintal, mikrotubulus kebanyakannya dijumpai sebagai struktur tiub berongga 13-helai (setiap helai disebut protofilamen). Juga, tubulin α dan β yang digunakan untuk membina mikrotubulus bukan sahaja bergantian, tetapi sebenarnya ditambahkan secara berpasangan. Kedua-α-tubulin dan β-tubulin mesti mengikat GTP untuk bergabung, tetapi setelah terikat, GTP yang terikat dengan α-tubulin tidak bergerak. Sebaliknya, GTP yang terikat dalam β-tubulin dapat dihidrolisis menjadi PDB. Αβ-dimer terikat PDB tidak akan ditambahkan ke mikrotubulus, jadi serupa dengan keadaan dengan ATP dan g-actin, jika tubulin mempunyai GDP yang terikat dengannya, ia mesti menukarnya dengan GTP terlebih dahulu sebelum dapat dipolimerisasi. Walaupun pertalian tubulin untuk GTP lebih tinggi daripada pertalian untuk KDNK, proses ini biasanya difasilitasi oleh GEF, atau faktor pertukaran nukleotida guanin. Oleh kerana bab transduksi isyarat akan ditunjukkan dengan lebih terperinci, pertukaran nukleotida jenis ini adalah mekanisme biasa untuk pengaktifan pelbagai jalur biokimia.

Sekali lagi seperti aktin, tubulin itu sendiri mempunyai aktiviti enzimatik, dan dari masa ke masa, aktiviti GTPase menghidrolisis GTP ke PDB dan fosfat. Ini mengubah keterikatan antara β-tubulin satu dimer dan α-tubulin dari dimer yang ditumpuknya kerana bentuk subunitnya berubah. Walaupun tidak secara langsung melonggarkan pegangan tubulin di sebelahnya, perubahan bentuk menyebabkan tekanan meningkat ketika bahagian mikrotubulus berusaha mendorong ke luar. Ini adalah asas sifat mikrotubulus yang dikenali sebagai ketidakstabilan dinamik. Sekiranya tidak ada yang menstabilkan mikrotubulus, sebahagian besarnya akan berantakan. Namun, selagi tubulin baru (yang akan terikat GTP) ditambahkan pada kadar yang cukup tinggi untuk menjaga bahagian mikrotubulus konfigurasi tekanan rendah "stabil" (disebut topi GTP) di atas GDP yang lebih tua bahagian, maka ia menstabilkan mikrotubulus keseluruhan. Apabila penambahan tubulin baru melambatkan, dan hanya terdapat penutup yang sangat kecil atau tidak ada, maka mikrotubulus mengalami malapetaka di mana sebahagian besar berpecah dengan cepat. Perhatikan bahawa ini adalah proses yang sangat berbeza daripada pemecahan oleh depolimerisasi, yang merupakan kehilangan secara beransur-ansur hanya beberapa subunit pada satu masa dari akhir mikrotubulus. Depolimerisasi juga berlaku, dan seperti aktin, ditentukan sebahagiannya oleh kepekatan relatif tubulin bebas dan mikrotubulus.

Dari sudut fizikal, mikrotubulus cukup kuat, tetapi tidak terlalu fleksibel. Suatu mikrofilamen akan lentur dan membengkokkan ketika daya ubah bentuk (bayangkan filamen berlabuh di hujung bawah berdiri lurus ke atas, dan sesuatu mendorong hujung ke satu sisi). Microtubule dalam keadaan yang sama hanya akan membengkok sedikit, tetapi pecah jika daya ubah bentuk mencukupi. Sudah tentu ada had fleksibiliti mikrofilamen dan akhirnya, ia juga akan pecah. Filamen pertengahan sedikit kurang fleksibel daripada mikrofilamen, tetapi dapat menahan kekuatan yang jauh lebih baik sama ada mikrofilamen atau mikrotubulus.


Sel (biologi)

The sel (dari bahasa Latin cella, yang bermaksud "bilik kecil" [1]) adalah unit struktur, fungsi, dan biologi asas bagi semua organisma yang diketahui. Sel adalah unit kehidupan terkecil, dan karenanya sering disebut sebagai "blok kehidupan". Kajian mengenai sel disebut biologi sel, biologi sel, atau sitologi.

Sel terdiri daripada sitoplasma yang tertutup dalam membran, yang mengandungi banyak biomolekul seperti protein dan asid nukleik. [2] Sebilangan besar sel tumbuhan dan haiwan hanya dapat dilihat di bawah mikroskop cahaya, dengan dimensi antara 1 dan 100 mikrometer. [3] Mikroskopi elektron memberikan resolusi yang jauh lebih tinggi yang menunjukkan struktur sel yang sangat terperinci. Organisma boleh dikelaskan sebagai uniselular (terdiri daripada satu sel seperti bakteria) atau multiselular (termasuk tumbuhan dan haiwan). [4] Kebanyakan organisma uniselular digolongkan sebagai mikroorganisma.

Jumlah sel dalam tumbuhan dan haiwan bervariasi dari spesies ke spesies yang dianggarkan bahawa manusia mengandung sekitar 40 trilion (4 × 10 13) sel. [a] [5] Otak manusia menyumbang sekitar 80 bilion sel ini. [6]

Sel dijumpai oleh Robert Hooke pada tahun 1665, yang menamakannya kerana kemiripannya dengan sel yang dihuni oleh biksu Kristian di sebuah biara. [7] [8] Teori sel, pertama kali dikembangkan pada tahun 1839 oleh Matthias Jakob Schleiden dan Theodor Schwann, menyatakan bahawa semua organisma terdiri daripada satu atau lebih sel, sel adalah unit asas struktur dan fungsi dalam semua organisma hidup, dan bahawa semua sel berasal dari sel yang ada sebelumnya. [9] Sel muncul di Bumi sekurang-kurangnya 3.5 bilion tahun yang lalu. [10] [11] [12]


Bab 12 Kitaran Sel

1. Terangkan bagaimana pembahagian sel berfungsi dalam pembiakan, pertumbuhan, dan pembaikan.

2. Huraikan organisasi struktur genom prokariotik dan eukariotik.

3. Huraikan peristiwa utama pembahagian sel yang membolehkan genom satu sel diteruskan kepada dua sel anak.

4. Terangkan bagaimana bilangan kromosom berubah sepanjang kitaran hidup manusia.

Kitaran Sel Mitotik

5. Senaraikan fasa kitaran sel dan terangkan urutan peristiwa yang berlaku pada setiap fasa.

6. Senaraikan fasa mitosis dan terangkan ciri peristiwa setiap fasa.

7. Kenali fasa mitosis dari gambar rajah dan mikrograf.

8. Lukis atau terangkan alat gelendong, termasuk sentrosom, mikrotubulus kinetochore, mikrotubulus nonkinetochore, aster, dan sentriol (dalam sel haiwan).

9. Huraikan perubahan ciri apa yang berlaku pada radas gelendong semasa setiap fasa mitosis.

10. Terangkan model semasa untuk pergerakan kromosom poleward dan pemanjangan paksi kutub sel.

11. Bandingkan sitokinesis pada haiwan dan tumbuhan.

12. Huraikan proses pembelahan binari dalam bakteria dan terangkan bagaimana mitosis eukariotik mungkin berkembang dari pembelahan binari.

Peraturan Kitaran Sel

13. Terangkan peranan pusat pemeriksaan, siklin, Cdk, dan MPF ​​dalam sistem kawalan kitaran sel.

14. Huraikan faktor dalaman dan luaran yang mempengaruhi sistem kawalan kitaran sel.

15. Terangkan bagaimana pembahagian sel sel barah yang tidak normal daripada kawalan kitaran sel normal.


12.4: Microtubules - Biologi

Twister RNA mewakili kelas ribozim semula jadi yang baru ditemui yang mempromosikan pembelahan tulang belakang RNA yang cepat. Walaupun terdapat banyak data teori, biokimia, dan struktur untuk beberapa anggota kelas twister, perbezaan pendapat mengenai seni bina dan mekanisme laman aktifnya telah muncul. Dari segi sejarah, ribut seperti itu mengenai perincian mekanistik biasanya berlaku sejurus selepas setiap kelas ribozim pembelahan diri baru dilaporkan, tetapi jalan ke hadapan ada untuk mencapai keadaan yang lebih tenang.

Proteolysis-Targeting Chimeras: Induced Protein Degradation sebagai Strategi Terapi

Sehingga baru-baru ini, satu-satunya cara untuk mengurangkan pemberian isyarat protein tertentu adalah dengan merobohkan sasaran oleh RNAi atau mengganggu isyarat dengan menghalang enzim atau reseptor dalam lata transduksi isyarat. Di sini, kami mengkaji kelas agen farmakologi molekul kecil yang muncul, yang disebut PROTAC, yang menyajikan pendekatan baru untuk mensasarkan protein secara khusus dan jalur isyarat masing-masing. Molekul-molekul heterobifungsi ini menggunakan mesin kawalan kualiti sel endogen dengan merekrutnya untuk mensasarkan protein untuk mendorong penurunannya.

Surat
Pemeriksaan Chemoproteomic Ligan Covalent Mendedahkan UBA5 Sebagai Sasaran Kanser Pankreas Novel
  • Allison M. Roberts,
  • David K. Miyamoto,
  • Tucker R. Huffman,
  • Leslie A. Bateman,
  • Ashley N. Ives,
  • David Akopian,
  • Martin J. Heslin,
  • Carlo M. Contreras,
  • Rogol Michael,
  • Christine F. Skibola, dan
  • Daniel K. Nomura*

Pemeriksaan genetik kimia dari perpustakaan molekul kecil telah menjadi strategi yang menjanjikan untuk menemui sebatian terapi yang unik dan baru. Walau bagaimanapun, mengenal pasti sasaran molekul plumbum yang muncul dari skrin ini tetap menjadi hambatan utama dalam memahami mekanisme tindakan sebatian ini. Di sini, kami telah menggabungkan penyaringan perpustakaan ligan kovalen berasaskan cysteine-reactive fragment dengan isotopic tandem orthogonal proteolysis-enabled protein-based profile profiling (isoTOP-ABPP) platform chemoproteomic untuk menghubungkan pasangan penemuan molekul kecil plumbum yang merosakkan pankreas patogenik barah dengan pengenalpastian kawasan panas yang boleh diminum untuk terapi barah yang berpotensi. Melalui pendekatan berpasangan ini, kami telah menemui ligan kovalen DKM 2–93 yang mengganggu kelangsungan hidup sel barah pankreas dan pertumbuhan tumor in vivo melalui pengubahsuaian katalitik sista dari ubiquitin seperti pengubah enzim pengaktifan 5 (UBA5), sehingga menghalang aktivitinya sebagai protein yang mengaktifkan protein seperti ubiquitin UFM1 kepada protein UFMylate. Kami menunjukkan bahawa UBA5 adalah sasaran terapi barah pankreas baru dan menunjukkan DKM 2–93 sebagai penghambat plumbum UBA5 yang relatif selektif. Hasil kami menggarisbawahi kegunaan penggabungan penyaringan perpustakaan ligan kovalen dengan platform isoTOP-ABPP untuk melombong proteome untuk kawasan panas yang boleh diberi ubat untuk terapi barah.

Argininosuccinate Synthase 1 adalah Metabolic Regulator of Colorectal Cancer Pathogenicity
  • Leslie A. Bateman,
  • Wan-Min Ku,
  • Martin J. Heslin,
  • Carlo M. Contreras,
  • Christine F. Skibola* , dan
  • Daniel K. Nomura*

Seperti banyak jenis barah, barah kolorektal mempunyai metabolisme yang tidak teratur yang meningkatkan ciri patogen mereka. Dalam kajian ini, kami menggunakan platform chemoproteomic profil protein berdasarkan aktiviti untuk profil enzim metabolik reaktif sistein yang diatur secara berlebihan pada tumor kolorektal primer manusia. Kami mengenal pasti argininosuccinate synthase 1 (ASS1) sebagai sasaran yang tidak diatur pada tumor kolorektal primer manusia dan menunjukkan bahawa perencatan farmakologi atau ablasi genetik ASS1 mengganggu patogenisitas kanser kolorektal. Dengan menggunakan profil metabolik, kami menunjukkan bahawa penghambatan ASS1 membawa kepada penurunan tahap metabolit onkogenik fumarat, yang menyebabkan penurunan metabolisme glikolitik yang menyokong patogenik sel barah kolorektal. Kami menunjukkan di sini bahawa perencat ASS1 mungkin mewakili pendekatan terapi baru untuk mengurangkan barah kolorektal dengan mengorbankan jalur isyarat metabolik dan metabolit kritikal dan menunjukkan kegunaan menggabungkan strategi kemoproteomik dan metabolom untuk memetakan pengatur metabolik barah baru.

Pencirian CYP115 Sebagai 3-Oksidase Gibberellin Menunjukkan bahawa Rhizobia Tertentu Dapat Menghasilkan Gibberellin A Bioaktif4

Fitohormon gibberellin (GA) dihasilkan bukan sahaja oleh tumbuhan tetapi juga oleh kulat dan bakteria. Pencirian sebelumnya dari opera biosintetik GA sitokrom P450 (CYP) yang terdapat dalam banyak rhizobia simbiotik, pembetulan nitrogen menyebabkan penjelasan biosintesis GA bakteria dan melibatkan GA9 sebagai produk akhir. Walau bagaimanapun, GA9 tidak menunjukkan aktiviti hormon / biologi dan mungkin memerlukan transformasi lebih lanjut untuk menimbulkan kesan di kilang inang kekacang. Beberapa rhizobia yang mengandungi operan GA juga memiliki CYP tambahan (CYP115), dan di sini kami menunjukkan bahawa ini bertindak sebagai GA 3-oksidase untuk menghasilkan GA4 bioaktif dari GA9. Ini adalah GA 3-oksidase pertama yang dikenal pasti untuk rhizobia, dan menyediakan skema yang lebih lengkap untuk biosintesis bioaktif GA pada bakteria. Selanjutnya, analisis filogenetik menunjukkan bahawa rhizobia memperoleh CYP115 secara bebas daripada operan inti GA, menambahkan kerumitan lebih lanjut kepada pemindahan gen mendatar enzim biosintetik GA di antara bakteria.

Mitokondria Cysteine ​​Desulfurase dan ISD11 dinyatakan dalam Escherichia coli Kompleks Hasil yang Mengandungi Protein Pembawa Acyl
  • Kai Cai,
  • Ronnie O. Frederick,
  • Marco Tonelli, dan
  • John L. Markley*

Mitokondria sistein desulfurase adalah komponen penting dalam mesin untuk biosintesis gugus besi-sulfur. Telah diketahui bahawa sistein desulfurase manusia yang aktif secara pemangkin secara in vitro dapat dibuat dengan mengekspresikan secara berlebihan dalam sel Escherichia coli dua komponen protein dari sistem ini, protein sistein desulfurase NFS1 dan protein tambahan ISD11. Kami melaporkan di sini bahawa penyediaan aktif ini mengandungi, tambahan, bentuk holo protein E. coli acyl carrier (Acp). Kami telah menentukan stoikiometri kompleks menjadi [Acp] 2: [ISD11] 2: [NFS1] 2. Protein pembawa asetil baru-baru ini didapati menjadi komponen penting dalam mesin biosintesis protein besi-sulfur di mitokondria, oleh kerana aktiviti [Acp] 2: [ISD11] 2: [NFS1] 2 dalam menyokong pemasangan kelompok besi-sulfur secara in vitro, nampaknya E. coli Acp dapat menggantikan homolog manusia.

Leptin-Tryptophan-Crosslinked Peptides Dihasilkan oleh Radikal SAM Enzim dalam Pathogenic Streptococci

Macrocycles mewakili kerangka struktur biasa dalam banyak peptida yang berlaku secara semula jadi. Beberapa strategi ada untuk makroklikisasi, dan enzim yang menggabungkannya sangat menarik, kerana mereka meningkatkan repertoar kami untuk membuat molekul kompleks. Kami baru-baru ini menemui reaksi siklisasi peptida baru yang melibatkan hubungan silang antara rantai sisi lisin dan triptofan yang dipasang oleh enzim SAM radikal. Di sini, kita mencirikan saudara-mara metalloenzim ini dari patogen Streptococcus agalactiae dan Streptococcus suis. Hasil kajian kami menunjukkan bahawa enzim yang sepadan, yang kami sebut AgaB dan SuiB, mengandungi banyak gugus [4Fe-4S] dan mengkatalisis pembentukan pautan silang Lys-Trp di substrat masing-masing. Analisis resolusi tinggi-MS dan 2D-NMR seterusnya terletak di lokasi makrocyclization. Lebih-lebih lagi, kami melaporkan bahawa AgaB dapat menerima substrat yang diubah suai yang mengandungi asid amino semula jadi atau tidak semula jadi. Selain memberikan pandangan mengenai mekanisme pengubahsuaian yang tidak biasa ini, persetujuan substrat AgaB dapat dieksploitasi untuk menghasilkan peptida makrosiklik yang pelbagai.

Probe Fluoresen membezakan antara Inhibisi Langkah Awal dan Akhir Biogenesis Lipopolysaccharide dalam Sel Sel
  • Eileen Moison,
  • Ran Xie,
  • Ge Zhang,
  • Matthew D. Lebar,
  • Timothy C. Meredith* , dan
  • Daniel Kahne*

Biogenesis lipopolysaccharide (LPS) dalam organisma Gram-negatif melibatkan biosintesisnya dalam sitoplasma dan seterusnya pengangkutan merentasi tiga petak selular ke permukaan sel. Kami mengembangkan probe pendarfluor yang membolehkan kami menentukan taburan ruang LPS di seluruh sel. Kami menunjukkan bahawa polymyxin B nonapeptide (PMBN) yang mengandungi dansyl fluorophore secara khusus mengikat LPS pada membran. Kami menunjukkan bahawa probe ini mengesan penurunan tahap LPS pada permukaan sel apabila biosintesis LPS dihambat pada langkah awal. Kami juga dapat mengesan pengumpulan LPS di lokasi subselular tertentu apabila pemasangan LPS disekat semasa pengangkutan, yang memungkinkan kita membezakan perencat yang mensasarkan tahap awal dan akhir biogenesis LPS.

Artikel
Isomer Struktural dan Epimer HPPH [3-Devinyl 3- pyropheophorbide-a]: Kesan Terapi Pengambilan dan Fotodinamik Kanser
  • Courtney Saenz,
  • Ravindra R. Cheruku,
  • Tymish Y. Ohulchanskyy,
  • Penny Joshi,
  • Walter A. Tabaczynski,
  • Joseph R. Missert,
  • Yihui Chen,
  • Paula Pera,
  • Erin Tracy,
  • Aimee Marko,
  • Daniel Rohrbach,
  • Ulas Sunar,
  • Heinz Baumann* , dan
  • Ravindra K. Pandey*

Struktur tetrapirol porfirin yang digunakan sebagai agen fotosentikasi dianggap menentukan pengambilan dan pengekalan oleh sel barah epitel malignan. Untuk menilai sumbangan keadaan pengoksidaan cincin individu untuk proses selular ini, bakterioklorofil a ditukar menjadi cincin "D" mengurangkan 3-devinyl-3- [1- (1-hexyloxy) ethyl] pyropheophorbide-a (HPPH) dan cincin isomer yang dikurangkan "B" yang sepadan (iso-HPPH). Analog asid karboksilik kedua-dua isomer cincin "B" dan cincin "D" menunjukkan pengumpulan beberapa kali lebih tinggi ke dalam mitokondria dan retikulum endoplasma oleh kultur primer sel barah paru-paru manusia dan kepala dan leher daripada analog ester metil yang sesuai yang melokalisasikan terutamanya ke vesikel berbutir dan pada tahap yang lebih rendah kepada mitokondria. Walau bagaimanapun, pengekalan sel-sel jangka panjang sebatian ini menunjukkan hubungan songsang dengan sel-sel tumor yang umumnya mengekalkan turunan metil-ester dengan lebih baik. Taburan in vivo dan pengambilan tumor dinilai dalam model isogenik tikus BALB / c yang membawa tumor Colon26 menggunakan analog berlabel 14C masing-masing. Kedua-dua derivatif asid karboksilik menunjukkan penyetempatan intraselular yang serupa dan penyembuhan tumor jangka panjang tanpa fototoksisiti kulit yang ketara. Tindakan tumor dimediasi PDT melibatkan kerosakan vaskular, yang disahkan oleh penurunan aliran darah dan penilaian imunohistokimia kerosakan pada endotelium vaskular. Stereoisomer HPPH (epimer) menunjukkan pengambilan yang sama (in vitro & amp in vivo), retensi intraselular dan photoreaction.

Mengenal pasti Residu Cysteine ​​Berfungsi di Mitokondria
  • Daniel W. Bak* ,
  • Mattia D. Pizzagalli, dan
  • Eranthie Weerapana*

Mitokondria adalah organel dinamik yang mengatur metabolisme oksidatif dan menengahi homeostasis redoks selular. Protein di dalam mitokondria terdedah kepada fluks besar di persekitaran redoks sekitarnya. Khususnya, residu sistein dalam protein mitokondria merasakan dan bertindak balas terhadap perubahan redoks ini melalui pengubahsuaian oksidatif kumpulan sistein thiol. Pengubahsuaian oksidatif ini mengakibatkan kehilangan reaktiviti sistein, yang dapat dipantau menggunakan probe kimia reaktif sistein dan spektrometri massa kuantitatif (MS). Analisis lisat sel yang dirawat dengan probe reaktif sistein memungkinkan pengenalan beratus-ratus residu sistein, namun, proteom mitokondria kurang diwakili (& lt10% peptida yang dikenal pasti), kerana rendahnya jumlah protein mitokondria dan penindasan isyarat MS peptida mitokondria oleh peptida sitosolik yang sangat banyak. Di sini, kami menerapkan protokol pengasingan dan pemurnian mitokondria untuk meningkatkan liputan protokol sistein mitokondria. Lebih 1500 residu sistein dari 50450 protein mitokondria dikenal pasti, sehingga memungkinkan untuk disoal siasat sejumlah sistein mitokondria yang belum pernah terjadi sebelumnya. Secara khusus, sistein mitokondria ini diberi peringkat berdasarkan kereaktifan untuk mengenal pasti sistein hiperaktif dengan peranan fungsi pemangkin dan pengatur yang berpotensi. Selanjutnya, analisis mitokondria yang terdedah kepada tekanan nitrosatif mendedahkan tapak protein S-nitrosasi pada protein mitokondria yang sebelumnya tidak dicirikan. Bersama-sama, strategi pengayaan sistein mitokondria yang disajikan di sini memungkinkan pencirian terperinci pengubahsuaian protein yang berlaku dalam mitokondria semasa (pato) aliran fisiologi di persekitaran redoks.

Protein SUV39H1 Lysine Methyltransferase Methylates Chromatin Protein Terlibat dalam Pembentukan Heterokromatin dan Penggabungan VDJ
  • Srikanth Kudithipudi,
  • Maren Kirstin Schuhmacher,
  • Adam Fiseha Kebede, dan
  • Albert Jeltsch*

SUV39H1 adalah metiltransferase H3K9 yang terlibat dalam pembentukan heterokromatin. Kami menyiasat profil kekhususan substratnya dan menunjukkan pengiktirafan residu H3 antara K4 dan G12 dengan bacaan R8 yang sangat spesifik. Profil kekhususan SUV39H1 berbeza dengan paralog SUV39H2, yang menunjukkan bahawa mereka boleh mempunyai substrat tambahan yang berbeza. Dengan menggunakan profil kekhususan, beberapa substrat calon SUV39H1 baru dikenal pasti. Kami memerhatikan metilasi 19 substrat novel pada tahap peptida dan enam daripadanya pada tahap protein. Metilasi RAG2, SET8, dan DOT1L disahkan dalam sel, yang semuanya mempunyai peranan penting dalam pengaturan kromatin. Metilasi SET8 secara alosterik merangsang aktiviti monometilasi H4K20 yang menghubungkan SUV39H1 ke generasi peningkatan tahap H4K20me3, satu lagi pengubahsuaian heterokromatik. Metilasi RAG2 mengubah lokalisasi subnuklearnya, menunjukkan bahawa SUV39H1 mungkin mengatur pengumpulan semula VDJ. Secara keseluruhan, hasil kami menunjukkan bahawa di luar generasi H3K9me3, SUV39H1 mempunyai peranan tambahan dalam biologi kromatin dengan rangsangan langsung pembentukan H4K20me3 dan pengaturan pengikatan kromatin RAG2.

Pengubahsuaian Perancah Antagonis Kovalen PPARγ Orthosterik Menghasilkan Penyekat Allosteric Dual-Site Peningkatan
  • Richard Brust,
  • Hua Lin,
  • Jakob Fuhrmann,
  • Alice Asteian,
  • Theodore M. Kamenecka, dan
  • Douglas J. Kojetin*

GW9662 dan T0070907 banyak digunakan antagonis tidak dapat dipulihkan yang tersedia secara komersil dari gamma reseptor diaktifkan proliferator peroksisom (PPARγ). Antagonis ini secara kovalen mengubah Cys285 yang terletak di dalam poket pengikat ligan ortosterik yang tertanam di domain pengikat ligan PPARγ dan digunakan untuk menyekat pengikatan ligan lain. Walau bagaimanapun, kami baru-baru ini mengenal pasti laman pengikat ligan alternatif / alosterik di PPARγ LBD yang pengikatan ligan tidak dihambat oleh antagonis kovalen ortosterik ini. Di sini, kami mengembangkan satu siri analog berdasarkan perancah antagonis kovalen ortosterik dengan tujuan untuk menghalang pengaktifan selular ortosterik dan allosterik PPARγ oleh MRL20, seorang agonis ortosterik yang juga mengikat ke laman web alosterik. Usaha kami menghasilkan pengenalan SR16832 (sebatian 22), yang berfungsi sebagai penghambat kovalen dua tempat transkripsi PPARγ oleh ligan pengikat PPARγ. Pemodelan molekul, analisis struktur spektroskopi protein NMR, dan ujian biokimia menunjukkan bahawa penghambatan pengaktifan allosterik berlaku sebahagiannya melalui pengembangan antagonis kovalen ortosterik 2-kloro-5-nitrobenzamidil ke laman alosterik, melemahkan pertalian ikatan ligan allosterik, dan mendorong konformasi perubahan tidak kompeten untuk pengaktifan PPARγ selular. Tambahan pula, SR16832 lebih baik menghalang pengikatan rosiglitazone, sebuah thiazolidinedione (TZD) yang lemah mengaktifkan PPARγ apabila dikotori dengan antagonis kovalen ortosterik, dan lebih baik dapat menghalang pengikatan ligan PPARγ endogen seperti asid docosahexaenoic (DHA) berbanding antagonis kovalen ortosterik. Sebatian seperti SR16832 mungkin merupakan alat kimia yang berguna untuk digunakan sebagai ortosterik bitopik dua tempat dan perencat kovalen allosterik ligan yang mengikat PPARγ.

Evolusi dan Pembahagian C7–Cyclitol Synthases di Prokaryotes dan Eukariota
  • Andrew R. Osborn,
  • Kelsey M. Kean,
  • Khaled M. Alseud,
  • Khaled H. Almabruk,
  • Shumpei Asamizu,
  • Janet A. Lee,
  • P. Andrew Karplus* , dan
  • Taifo Mahmud*

2-Epi-5-epi-valiolone synthase (EEVS), C7-sugar phosphate cyclase (SPC) homologous to 3-dehydroquinate synthase (DHQS), ditemui semasa kajian mengenai biosintesis keluarga C7N-aminocyclitol produk semula jadi. EEVS pada awalnya dianggap hanya terdapat pada aktinomycetes tertentu, tetapi analisis urutan genom menunjukkan bahawa ia diedarkan secara meluas di prokariota dan eukariota, termasuk vertebrata. SPC lain, desmethyl-4-deoxygadusol synthase (DDGS), kemudian didapati terlibat dalam biosintesis sebatian pelindung matahari asid amino seperti mikrosorin. Anotasi pangkalan data semasa agak tidak boleh dipercayai, dengan banyak EEVS dilaporkan sebagai DHQS, dan kebanyakan DDGS dilaporkan sebagai EEVS, DHQS, atau hanya protein hipotesis. Di sini, kami mengenal pasti ciri urutan yang berguna untuk membezakan enzim ini, melaporkan struktur kristal DDGS perwakilan yang menunjukkan persamaan tinggi enzim EEVS dan DDGS, mengenal pasti perbezaan tapak aktif yang terkenal, dan menunjukkan pentingnya dua residu tapak aktif ini untuk pemangkin oleh mutasi titik. Selanjutnya, kami secara fungsional menggambarkan dua wakil dari clade yang berbeza dengan jarak EEVS dan kumpulan DDGS yang diketahui dan menunjukkan mereka sebagai EEVS yang sahih. Lebih-lebih lagi, kami mendokumentasikan dan membincangkan pengedaran gen yang mengekod EEVS dan DDGS dalam pelbagai prokariota dan eukariota, termasuk bakteria patogen, simbion tumbuhan, bakteria penentu nitrogen, myxobacteria, cyanobacteria, fungi, stramenopiles, dan haiwan, menunjukkan peranan biologi berpotensi luas mereka secara semula jadi.

Penentu Kekhususan Urutan BH3 untuk Gangguan Kompleks Bcl-xL / cBid dalam Membran

Protein prosurvival Bcl-2 menunjukkan corak interaksi tertentu dengan protein BH3 sahaja yang menentukan pergantungan sel pada tekanan apoptosis. Kekhususan ini sangat penting untuk pengembangan mimetik BH3, kelas molekul antikanker berdasarkan domain BH3 dengan aktiviti yang menjanjikan dalam ujian klinikal. Walaupun pembentukan kompleks berlaku terutamanya pada membran luar mitokondria, kebanyakan kajian setakat ini menangani interaksi antara peptida BH3 dan protein Bcl-2 terpotong dalam larutan. Akibatnya, pemahaman kuantitatif penentu kekhususan urutan peptida BH3 di persekitaran membran hilang. Di sini, kita mengatasi masalah ini dengan secara sistematik mengukur kemampuan peptida BH3 untuk bersaing untuk kompleks antara cBid dan Bcl-xL dalam vesikel unilamellar gergasi dan membandingkannya dengan larutan dan mitokondria. Kami menunjukkan bahawa peptida BH3 yang berasal dari Hrk, Bim, Bid, dan Bad adalah yang paling berkesan dalam mengganggu kompleks cBid / Bcl-xL dalam membran, yang berkorelasi dengan aktiviti mereka dalam mitokondria. Penemuan kami menyokong penargetan membran molekul kecil yang mengikat protein Bcl-2 sebagai strategi untuk meningkatkan kecekapannya.

Kawalan Penyusunan Semula Foto-Claisen Yang Tidak Biasa di Coumarin Caged Tamoxifen melalui Extended Spacer
  • Pamela T. Wong,
  • Edward W. Roberts,
  • Shengzhuang Tang,
  • Jhindan Mukherjee,
  • Jayme Cannon,
  • Alyssa J. Nip,
  • Kaitlin Corbin,
  • Matthew F. Krummel* , dan
  • Seok Ki Choi*

Penggunaan molekul coumarin telah didokumentasikan dengan baik dalam banyak aplikasi pemotretan termasuk untuk pengendalian spatiotemporal aktiviti rekombinase reseptor Cre-estrogen (Cre-ERT2). Dalam artikel ini, kami melaporkan bahawa 4-hydroxytamoxifen (4OHT) dikurung dengan coumarin melalui hubungan ether konvensional menyebabkan penyusunan semula foto-Claisen yang tidak dijangka yang secara signifikan bersaing dengan pembebasan 4OHT percuma. Asas bagi reaksi yang tidak diingini ini tampaknya berkaitan dengan struktur kumarin dan mekanisme radikal berdasarkan penguraian, kerana ia tidak berlaku pada sangkar ortho-nitrobenzil (ONB) 4OHT yang sebaliknya dihubungkan dengan cara yang sama. Dalam usaha untuk melakukan pengoptimuman reka bentuk, kami memperkenalkan penghubung self-immolative lebih lama daripada penghubung eter dan mengenal pasti penghubung optimum yang membolehkan pelepasan 4OHT cepat oleh mekanisme penyerapan foton tunggal dan dua foton. Keupayaan konstruk ini untuk secara aktif mengawal pengubahsuaian gen yang dimediasi Cre-ERT2 disiasat dalam fibroblas embrio tikus (MEF) di mana ekspresi penggabungan gen yang bergantung kepada wartawan protein pendarfluor hijau (GFP) dikendalikan oleh pelepasan 4OHT dan diukur dengan mikroskopi pendarfluor konfokal dan sitometri aliran. Ringkasnya, kami melaporkan implikasi penyusunan foto-Claisen ini dalam sebatian sangkar kumarin dan menunjukkan strategi penghubung rasional untuk menangani reaksi sampingan yang tidak diingini ini.

Aktiviti JmjC Histone Lysine Demethylase KDM4A Sangat Sensitif terhadap Kepekatan Oksigen
  • Rebecca L Hancock,
  • Norma Masson,
  • Kate Dunne,
  • Emily Flashman* , dan
  • Akane Kawamura*

The JmjC histone lysine demethylases (KDMs) adalah pengatur epigenetik yang terlibat dalam penyingkiran kumpulan metil dari residu lisil yang diubahsuai selepas terjemahan dalam ekor histon, memodulasi transkripsi gen. Enzim ini memerlukan oksigen molekul untuk aktiviti pemangkin dan, kerana oksigenase yang bergantung pada 2-oxoglutarate (2OG), berkaitan dengan oksigen sel yang merasakan hidroksilase HIF PHD2 dan FIH. Kajian terbaru menunjukkan bahawa aktiviti beberapa KDM, termasuk KDM4E yang dikodkan pseudogene, mungkin sensitif terhadap perubahan kepekatan oksigen. Di sini, kami melaporkan analisis terperinci mengenai kesan ketersediaan oksigen pada aktiviti anggota subkeluarga KDM4 KDM4A, yang penting menunjukkan tahap kepekaan O2 yang tinggi baik dengan protein terpencil dan sel. Analisis kinetik enzim rekombinan menunjukkan KMapp (O2) tinggi 173 ± 23 μM, menunjukkan bahawa aktiviti enzim dapat bertindak balas secara sensitif terhadap penurunan kepekatan oksigen. Selanjutnya, eksperimen imunofluoresensi pada sel U2OS secara bersyarat terlalu banyak menyatakan KDM4A menunjukkan bahawa aktiviti sel KDM4A terhadap substrat utamanya, H3K9me3, menunjukkan tindak balas berperingkat terhadap penurunan kepekatan oksigen sejajar dengan data yang diperoleh menggunakan protein terpencil. Hasil ini menunjukkan bahawa KDM4A memiliki potensi untuk bertindak sebagai sensor oksigen dalam konteks modifikasi kromatin, dengan kemungkinan implikasi terhadap peraturan epigenetik pada keadaan penyakit hipoksia. Yang penting, hubungan ini antara kepekaan oksigen dari aktiviti pemangkin KDM4A dalam ujian biokimia dan selular menunjukkan kegunaan kajian biokimia dalam memahami faktor-faktor yang menyumbang kepada pelbagai fungsi biologi dan pelbagai aktiviti oksigenase 2OG.

O-GlcNAcylation of a-Synuclein at Serine 87 Mengurangkan Agregasi tanpa Mempengaruhi Membran Binding
  • Yuka E. Lewis,
  • Ana Galesic,
  • Paul M. Levine,
  • Cesar A. De Leon,
  • Natalie Lamiri,
  • Caroline K. Brennan, dan
  • Matthew R. Pratt*

Penggabungan protein yang berkaitan dengan penyakit neurodegeneratif dapat dipengaruhi oleh banyak faktor, termasuk pelbagai modifikasi pasca-terjemahan. Salah satu pengubahsuaian tersebut, O-GlcNAcylation, telah dijumpai pada sebilangan protein agregasi ini, termasuk α-synuclein, protein utama yang memainkan peranan penyebab dalam synucleinopathies seperti penyakit Parkinson. Kami sebelum ini menggunakan kimia protein sintetik untuk menyediakan α-synuclein yang mengandungi pengubahsuaian O-GlcNAc homogen pada threonine 72 dan menunjukkan bahawa pengubahsuaian ini menghalang pengagregatan protein. Walau bagaimanapun, kesan lapan laman web O-GlcNAcylation lain yang telah dikenal pasti tidak diketahui. Di sini, kami menggunakan strategi sintetik yang serupa untuk menyiasat akibat pengubahsuaian ini di salah satu laman web ini, serine 87. Kami menunjukkan bahawa O-GlcNAcylation di laman web ini juga menghalang agregasi α-synuclein tetapi pada tahap yang lebih rendah daripada pengubahsuaian yang sama di threonine 72. Walau bagaimanapun, kami juga mendapati bahawa pengubahsuaian ini tidak mempengaruhi sifat mengikat membran α-synuclein, yang membezakannya dari fosforilasi di laman web yang sama. Hasil ini menyokong pengembangan terapi yang dapat meningkatkan O-GlcNAcylation a-synuclein untuk melambatkan perkembangan penyakit Parkinson.

Highly Potent Cell-Permeable and Impermeable NanoLuc Luciferase Inhibitors
  • Joel R. Walker* ,
  • Mary P. Hall ,
  • Chad A. Zimprich ,
  • Matthew B. Robers ,
  • Sarah J. Duellman ,
  • Thomas Machleidt,
  • Jacquelynn Rodriguez , and
  • Wenhui Zhou

Novel engineered NanoLuc (Nluc) luciferase being smaller, brighter, and superior to traditional firefly (Fluc) or Renilla (Rluc) provides a great opportunity for the development of numerous biological, biomedical, clinical, and food and environmental safety applications. This new platform created an urgent need for Nluc inhibitors that could allow selective bioluminescent suppression and multiplexing compatibility with existing luminescence or fluorescence assays. Starting from thienopyrrole carboxylate 1, a hit from a 42 000 PubChem compound library with a low micromolar IC50 against Nluc, we derivatized four different structural fragments to discover a family of potent, single digit nanomolar, cell permeable inhibitors. Further elaboration revealed a channel that allowed access to the external Nluc surface, resulting in a series of highly potent cell impermeable Nluc inhibitors with negatively charged groups likely extending to the protein surface. The permeability was evaluated by comparing EC50 shifts calculated from both live and lysed cells expressing Nluc cytosolically. Luminescence imaging further confirmed that cell permeable compounds inhibit both intracellular and extracellular Nluc, whereas less permeable compounds differentially inhibit extracellular Nluc and Nluc on the cell surface. The compounds displayed little to no toxicity to cells and high luciferase specificity, showing no activity against firefly luciferase or even the closely related NanoBit system. Looking forward, the structural motifs used to gain access to the Nluc surface can also be appended with other functional groups, and therefore interesting opportunities for developing assays based on relief-of-inhibition can be envisioned.

Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity
  • Geng-Yuan Chen ,
  • You Jung Kang ,
  • A. Sophia Gayek ,
  • Wiphu Youyen ,
  • Erkan Tüzel ,
  • Ryoma Ohi , and
  • William O. Hancock*

To uncover their contrasting mechanisms, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 “braking” dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM KI. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.

A Fluorescent Hsp90 Probe Demonstrates the Unique Association between Extracellular Hsp90 and Malignancy dalam Vivo
  • Lauren B. Crowe ,
  • Philip F. Hughes ,
  • David A. Alcorta ,
  • Takuya Osada ,
  • Aaron P. Smith ,
  • Juliane Totzke ,
  • David R. Loiselle ,
  • Isaac D. Lutz ,
  • Madhusudhana Gargesha ,
  • Debasish Roy ,
  • Jose Roques ,
  • David Darr ,
  • H. Kim Lyerly ,
  • Neil L. Spector , and
  • Timothy A.J. Haystead*

Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is correlated with malignancy. Development of small molecule probes that can detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a cell impermeable far-red fluorophore-tagged Hsp90 inhibitor to target eHsp90 in vivo. High resolution confocal and lattice light sheet microscopy show that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is actively internalized. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by overexpressing p110HER2. Whole body cryoslicing, imaging, and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression and internalization is a phenomenon unique to tumor cells in vivo and may provide an “Achilles heel” for the early diagnosis of metastatic disease and targeted drug delivery.

Iron Release from the Siderophore Pyoverdine in Pseudomonas aeruginosa Involves Three New Actors: FpvC, FpvG, and FpvH
  • Géraldine Ganne ,
  • Karl Brillet ,
  • Beata Basta ,
  • Béatrice Roche ,
  • Françoise Hoegy ,
  • Véronique Gasser , and
  • Isabelle J. Schalk*

Siderophores are iron chelators produced by bacteria to access iron, an essential nutriment. Pyoverdine (PVDI), the major siderophore produced by Pseudomonas aeruginosa PAO1, consists of a fluorescent chromophore linked to an octapeptide. The ferric form of PVDI is transported from the extracellular environment into the periplasm by the outer membrane transporter, FpvA. Iron is then released from the siderophore in the periplasm by a mechanism that does not involve chemical modification of the chelator but an iron reduction step. Here, we followed the kinetics of iron release from PVDI, in vitro and in living cells, by monitoring its fluorescence (as apo PVDI is fluorescent, whereas PVDI-Fe(III) is not). Deletion of the inner membrane proteins fpvG (PA2403) and fpvH (PA2404) affected 55Fe uptake via PVDI and completely abolished PVDI-Fe dissociation, indicating that these two proteins are involved in iron acquisition via this siderophore. PVDI-Fe dissociation studies, using an in vitro assay, showed that iron release from this siderophore requires the presence of an iron reducer (DTT) and an iron chelator (ferrozine). In this assay, DTT could be replaced by the inner membrane protein, FpvG, and ferrozine by the periplasmic protein, FpvC, suggesting that FpvG acts as a reductase and FpvC as an Fe2+ chelator in the process of PVDI-Fe dissociation in the periplasm of P. aeruginosa cells. This mechanism of iron release from PVDI is atypical among Gram-negative bacteria but seems to be conserved among Pseudomonads.

The GCaMP-R Family of Genetically Encoded Ratiometric Calcium Indicators
  • Jung-Hwa Cho ,
  • Carter J. Swanson ,
  • Jeannie Chen ,
  • Ang Li ,
  • Lisa G. Lippert ,
  • Shannon E. Boye ,
  • Kasey Rose ,
  • Sivaraj Sivaramakrishnan ,
  • Cheng-Ming Chuong , and
  • Robert H. Chow*

We report on GCaMP-Rs, a new family of genetically encoded ratiometric calcium indicators that extend the virtues of the GCaMP proteins to ratiometric measurements. We have engineered a tandem construct of calcium-dependent GCaMP and calcium-independent mCherry fluorescent proteins. The tandem design assures that the two proteins localize in the same cellular compartment(s) and facilitates pixelwise ratiometric measurements however, Förster resonance energy transfer (FRET) between the fluorophores reduces brightness of the sensor by up to half (depending on the GCaMP variant). To eliminate FRET, we introduced a rigid α-helix, the ER/K helix, between GCaMP and mCherry. Avoiding FRET significantly increases the brightness (notably, even at low calcium concentrations), the signal-to-noise ratio, and the dynamic range.

Discovery and Characterization of a Potent and Specific Peptide Ligand Targeting Endothelial Progenitor Cells and Endothelial Cells for Tissue Regeneration
  • Dake Hao ,
  • Wenwu Xiao ,
  • Ruiwu Liu ,
  • Priyadarsini Kumar ,
  • Yuanpei Li ,
  • Ping Zhou ,
  • Fuzheng Guo ,
  • Diana L. Farmer ,
  • Kit S. Lam ,
  • Fengshan Wang* , dan
  • Aijun Wang*

Endothelial progenitor cells (EPCs) and endothelial cells (ECs) play a vital role in endothelialization and vascularization for tissue regeneration. Various EPC/EC targeting biomolecules have been investigated to improve tissue regeneration with limited success often due to their limited functional specificity and structural stability. One-bead one-compound (OBOC) combinatorial technology is an ultrahigh throughput chemical library synthesis and screening method suitable for ligand discovery against a wide range of biological targets, such as integrins. In this study, using primary human EPCs/ECs as living probes, we identified an αvβ3 integrin ligand LXW7 discovered by OBOC combinatorial technology as a potent and specific EPC/EC targeting ligand. LXW7 overcomes the major barriers of other functional biomolecules that have previously been used to improve vascularization for tissue regeneration and possesses optimal stability, EPC/EC specificity, and functionality. LXW7 is a disulfide cyclic octa-peptide (cGRGDdvc) containing unnatural amino acids flanking both sides of the main functional motif therefore it will be more resistant to proteolysis and more stable in vivo compared to linear peptides and peptides consisting of only natural amino acids. Compared with the conventional αvβ3 integrin ligand GRGD peptide, LXW7 showed stronger binding affinity to primary EPCs/ECs but weaker binding to platelets and no binding to THP-1 monocytes. In addition, ECs bound to the LXW7 treated culture surface exhibited enhanced biological functions such as proliferation, likely due to increased phosphorylation of VEGF receptor 2 (VEGF-R2) and activation of mitogen-activated protein kinase (MAPK) ERK1/2. Surface modification of electrospun microfibrous PLLA/PCL biomaterial scaffolds with LXW7 via Click chemistry resulted in significantly improved endothelial coverage. LXW7 and its derivatives hold great promise for EPC/EC recruitment and delivery and can be widely applied to functionalize various biological and medical materials to improve endothelialization and vascularization for tissue regeneration applications.

Fluorescent Hexose Conjugates Establish Stringent Stereochemical Requirement by GLUT5 for Recognition and Transport of Monosaccharides
  • Olivier-Mohamad Soueidan ,
  • Thomas W. Scully ,
  • Jatinder Kaur ,
  • Rashmi Panigrahi ,
  • Alexandr Belovodskiy ,
  • Victor Do ,
  • Carson D. Matier ,
  • M. Joanne Lemieux ,
  • Frank Wuest ,
  • Chris Cheeseman* , dan
  • F. G. West*

The specificity characteristics of transporters can be exploited for the development of novel diagnostic therapeutic probes. The facilitated hexose transporter family (GLUTs) has a distinct set of preferences for monosaccharide substrates, and while some are expressed ubiquitously (e.g., GLUT1), others are quite tissue specific (e.g., GLUT5, which is overexpressed in some breast cancer tissues). While these differences have enabled the development of new molecular probes based upon hexose- and tissue-selective uptake, substrate design for compounds targeting these GLUT transporters has been encumbered by a limited understanding of the molecular interactions at play in hexose binding and transport. Four new fluorescently labeled hexose derivatives have been prepared, and their transport characteristics were examined in two breast cancer cell lines expressing mainly GLUTs 1, 2, and 5. Our results demonstrate, for the first time, a stringent stereochemical requirement for recognition and transport by GLUT5. 6-NBDF, in which all substituents are in the d-fructose configuration, is taken up rapidly into both cell lines via GLUT5. On the other hand, inversion of a single stereocenter at C-3 (6-NBDP), C-4 (6-NBDT), or C-5 (6-NDBS) results in selective transport via GLUT1. An in silico docking study employing the recently published GLUT5 crystal structure confirms this stereochemical dependence. This work provides insight into hexose-GLUT interactions at the molecular level and will facilitate structure-based design of novel substrates targeting individual members of the GLUT family and forms the basis of new cancer imaging or therapeutic agents.

The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase
  • Elise A. Span ,
  • Daniel L. M. Suess ,
  • Marc C. Deller ,
  • R. David Britt , and
  • Michael A. Marletta*

Polysaccharide monooxygenases (PMOs) are secreted metalloenzymes that catalyze the oxidative degradation of polysaccharides in a copper-, oxygen-, and reductant-dependent manner. Cellulose-active fungal PMOs degrade cellulosic substrates to be utilized as a carbon source for fungal growth. To gain insight into the PMO mechanism, the role of conserved residues in the copper coordination sphere was investigated. Here, we report active-site hydrogen-bonding motifs in the secondary copper coordination sphere of MtPMO3*, a C1-oxidizing PMO from the ascomycete fungus Myceliophthora thermophila. A series of point substitutions that disrupt this conserved network are used to interrogate its function. Activity assays, in conjunction with EPR spectroscopy, demonstrate that residues H161 and Q167 are involved in stabilizing bound oxygen, and H161 appears to play a role in proton transfer. Additionally, Q167 increases the ligand donor strength of Y169 to the copper via a hydrogen-bonding interaction. Altogether, H161 and Q167 are important for oxygen activation, and the results are suggestive of a copper–oxyl active intermediate.

NMR and Molecular Recognition of N-Glycans: Remote Modifications of the Saccharide Chain Modulate Binding Features
  • Ana Gimeno ,
  • Niels-Christian Reichardt ,
  • F. Javier Cañada ,
  • Lukas Perkams ,
  • Carlo Unverzagt ,
  • Jesús Jiménez-Barbero* , dan
  • Ana Ardá*

Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate–protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore their importance for the development of biomedical applications.

Ion Mobility-Mass Spectrometry Reveals a Dipeptide That Acts as a Molecular Chaperone for Amyloid β
  • Molly T. Soper-Hopper ,
  • Joseph D. Eschweiler , and
  • Brandon T. Ruotolo*

Previously, we discovered and structurally characterized a complex between amyloid β 1–40 and the neuropeptide leucine enkephalin. This work identified leucine enkephalin as a potentially useful starting point for the discovery of peptide-related biotherapeutics for Alzheimer’s disease. In order to better understand such complexes that are formed in vitro, we describe here the analysis of a series of site-directed amino acid substitution variants of both peptides, covering the leucine enkephalin sequence in its entirety and a large number of selected residues of amyloid β 1–40 (residues: D1, E3, F4, R5, H6, Y10, E11, H13, H14, Q15, K16, E22, K28, and V40). Ion mobility–mass spectrometry measurements and molecular dynamics simulations reveal that the hydrophobic C-terminus of leucine enkephalin (Phe-Leu, FL) is crucial for the formation of peptide complexes. As such, we explore here the interaction of the dipeptide FL with both wildtype and variant forms of amyloid β in order to structurally characterize the complexes formed. We find that FL binds preferentially to amyloid β oligomers and attaches to amyloid β within the region between its N-terminus and its hydrophobic core, most specifically at residues Y10 and Q15. We further show that FL is able to prevent fibril formation.

A Near-Infrared, Wavelength-Shiftable, Turn-on Fluorescent Probe for the Detection and Imaging of Cancer Tumor Cells
  • Zhenhua Shen ,
  • Bijeta Prasai ,
  • Yuko Nakamura ,
  • Hisataka Kobayashi ,
  • Milcah S. Jackson , and
  • Robin L. McCarley*

Fast, selective, and noninvasive reporting of intracellular cancer-associated events and species will lead to a better understanding of tumorigenesis at the molecular level and development of precision medicine approaches in oncology. Overexpressed reductase presence in solid tumor cells is key to cancer progression and protection of those diseased cells from the oxidative effects of therapeutics meant to kill them. Human NAD(P)H:quinone oxidoreductase isozyme I (hNQO1), a cytoprotective 2-electron-specific reductase found at unusually high activity levels in cancer cells of multiple origins, has attracted significant attention due to its major role in metastatic pathways and its link to low survival rates in patients, as well as its ability to effectively activate quinone-based, anticancer drugs. Accurate assessment of hNQO1 activities in living tumor models and ready differentiation of metastases from healthy tissue by fluorescent light-based protocols requires creation of hNQO1-responsive, near-infrared probes that offer deep tissue penetration and low background fluorescence. Herein, we disclose a quinone-trigger-based, near-infrared probe whose fluorescence is effectively turned on several hundred-fold through highly selective reduction of the quinone trigger group by hNQO1, with unprecedented, catalytically efficient formation of a fluorescent reporter. hNQO1 activity-specific production of a fluorescence signal in two-dimensional cultures of respiring human cancer cells that harbor the reductase enzyme allows for their quick (30 min) high-integrity recognition. The characteristics of the near-infrared probe make possible the imaging of clinically relevant three-dimensional colorectal tumor models possessing spatially heterogeneous hNQO1 activities and provide for fluorescence-assisted identification of submillimeter dimension metastases in a preclinical mouse model of human ovarian serous adenocarcinoma.

Mapping Novel Metabolic Nodes Targeted by Anti-Cancer Drugs that Impair Triple-Negative Breast Cancer Pathogenicity
  • Lindsay S. Roberts ,
  • Peter Yan ,
  • Leslie A. Bateman , and
  • Daniel K. Nomura*

Triple-negative breast cancers (TNBCs) are estrogen receptor, progesterone receptor, and HER2 receptor-negative subtypes of breast cancers that show the worst prognoses and lack targeted therapies. Here, we have coupled the screening of ∼400 anticancer agents that are under development or in the clinic with chemoproteomic and metabolomic profiling to identify novel metabolic mechanisms for agents that impair TNBC pathogenicity. We identify 20 anticancer compounds that significantly impaired cell survival across multiple types of TNBC cells. Among these 20 leads, the phytoestrogenic natural product licochalcone A was of interest, since TNBCs are unresponsive to estrogenic therapies, indicating that licochalcone A was likely acting through another target. Using chemoproteomic profiling approaches, we reveal that licochalcone A impairs TNBC pathogenicity, not through modulating estrogen receptor activity but rather through inhibiting prostaglandin reductase 1, a metabolic enzyme involved in leukotriene B4 inactivation. We also more broadly performed metabolomic profiling to map additional metabolic mechanisms of compounds that impair TNBC pathogenicity. Overlaying lipidomic profiling with drug responses, we find that deubiquitinase inhibitors cause dramatic elevations in acyl carnitine levels, which impair mitochondrial respiration and contribute to TNBC pathogenic impairments. We thus put forth two unique metabolic nodes that are targeted by drugs or drug candidates that impair TNBC pathogenicity. Our results also showcase the utility of coupling drug screens with chemoproteomic and metabolomic profiling to uncover unique metabolic drivers of TNBC pathogenicity.

Structure–Activity Relationships of the Competence Stimulating Peptides (CSPs) in Streptococcus pneumoniae Reveal Motifs Critical for Intra-group and Cross-group ComD Receptor Activation
  • Yifang Yang ,
  • Bimal Koirala ,
  • Lucia A. Sanchez ,
  • Naiya R. Phillips ,
  • Sally R. Hamry , and
  • Yftah Tal-Gan*

Streptococcus pneumoniae is a highly recombinogenic human pathogen that utilizes the competence stimulating peptide (CSP)-based quorum sensing (QS) circuitry to acquire antibiotic resistance genes from the environment and initiate its attack on the human host. Modulation of QS in this bacterium, either inhibition or activation, can therefore be used to attenuate S. pneumoniae infectivity and slow down pneumococcal resistance development. In this study, we set to determine the molecular mechanism that drives CSP:receptor binding and identify CSP-based QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in the two major CSP signals (CSP1 and CSP2) and assessed the ability of the mutated analogs to modulate QS against both cognate and noncognate ComD receptors. We then evaluated the overall 3D structures of these analogs using circular dichroism (CD) to correlate between the structure and function of these peptides. Our CD analysis revealed a strong correlation between α-helicity and bioactivity for both specificity groups (CSP1 and CSP2). Furthermore, we identified the first pan-group QS activator and the most potent group-II QS inhibitor to date. These chemical probes can be used to study the role of QS in S. pneumoniae and as scaffolds for the design of QS-based anti-infective therapeutics against S. pneumoniae infections.

Glycation of Lysozyme by Glycolaldehyde Provides New Mechanistic Insights in Diabetes-Related Protein Aggregation
  • Laura Mariño ,
  • Carlos Andrés Maya-Aguirre ,
  • Kris Pauwels ,
  • Bartolomé Vilanova ,
  • Joaquin Ortega-Castro ,
  • Juan Frau ,
  • Josefa Donoso , and
  • Miquel Adrover*

Glycation occurs in vivo as a result of the nonenzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA, or lipids. Protein glycation causes loss-of-function and, consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question of whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the micromolar range). At low HEWL concentration, non-cross-linking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have little impact on HEWL surface hydrophobicity and, therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shifts toward the formation of intermolecular cross-links, which triggers a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic interactions. Additionally, their comparison constitutes the first experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.


Carbocyanine Dyes

Short-Chain Carbocyanine Dyes

Terasaki and co-workers used the short-chain carbocyanine DiOC6(3) (D273) to visualize the ER in both live and aldehyde-fixed cells. This dye and the similar DiOC5(3) have since been used extensively to study structural interactions and dynamics of the ER in neurons, yeast and onion epidermis, and to examine the morphological relationships between the ER, mitochondria, intermediate filaments and microtubules in various cell types. DiOC6(3) and DiOC5(3) pass through the plasma membrane and stain intracellular membranes with a fluorescein-like fluorescence ER membranes can easily be distinguished by their characteristic morphology. Caution must be exercised, however, in using the carbocyanines as probes for the ER. It has been reported that ER staining with DiOC6(3) does not occur until the mitochondria round up and lose the fluorochrome. Rhodamine 6G and the hexyl ester of rhodamine B (R634, R648MP Probes for Mitochondria—Section 12.2) appear to stain like DiOC6(3), except they are apparently less toxic and they fluoresce orange, providing possibilities for multicolor labeling. When used at very low concentrations, these slightly lipophilic rhodamine dyes tend to stain only mitochondria of live cells.

Long-Chain Carbocyanine Dyes

Terasaki and Jaffe have used the long-chain carbocyanines DiIC16(3) and DiIC18(3) (D384, D282) to label ER membranes. They achieved selective labeling of the ER by microinjecting a saturated solution of DiI in oil into sea urchin eggs. This method has been successful in several other egg types but was not effective in molluscan or arthropod axons. As noted in the discussion of dialkylcarbocyanine and dialkylaminostyryl probes in Dialkylcarbocyanine and Dialkylaminostyryl Probes—Section 13.4, DiI diffuses only in continuous membranes.


PENGENALAN

Important advances in our understanding of the cytoskeleton have been made by direct observations of living cells following microinjection with fluorescent derivatives of cytoskeletal proteins (Desai and Mitchison, 1997). More recently, however, the ability to express cloned proteins containing a green fluorescent protein (GFP) tag has become the method of choice for dynamic analysis of the cytoskeleton (Chalfie et al., 1994). GFP technology offers several significant advantages over the previous technology: it is not necessary to microinject cells, nor is it necessary to biochemically purify and fluorescently modify the protein of interest. However, there are also limitations to GFP technology: addition of GFP (238 amino acids) to the C or N terminus of the target protein can potentially interfere with protein function. In this regard, it is important to demonstrate that the chimeric protein retains its normal characteristics. In addition, overexpression of any protein can potentially interfere with cellular functions. This latter limitation can be overcome by the use of inducible promoters in the plasmid construct or by establishing permanent cell lines with the desired level of expression of the chimera.

To date, the dynamics of several cytoskeletal proteins have been examined using GFP technology. For example, transformation of yeast with a GFP-actin construct was used to document the motion of cortical actin patches (Doyle and Botstein, 1996). Although the GFP-actin did incorporate into dynamic actin-containing structures in the cells, the construct was not able to complement an actin null mutant (Doyle and Botstein, 1996). The major yeast tubulin gene tub1 has also been tagged with GFP and this construct rescues a tub1mutant (Straight et al., 1997). Importantly, observation of mitosis in yeast transformed with GFP-tub1 provides strong evidence that spindle microtubules can undergo normal dynamic behavior in the expressing cells (Straight et al., 1997). In other experiments, a fusion of GFP to the amino terminus of Tub1p did not complement a tub1 deletion mutation, but yeast cells expressing a mixture of GFP-tagged and wild-type tubulin grew at normal rates (Maddox et al., 1999). The dynamic behavior of individual microtubules has also been examined in yeast expressing an amino terminal fusion of GFP to a different yeast tubulin gene,tub3. The results show that yeast microtubules undergo dynamic instability behavior that is cell cycle regulated (Carminati and Stearns, 1997 Tirnauer et al., 1999). In these experiments, addition of GFP to the amino terminus of tub3, but not to the carboxy terminus, was able to complement atub3 null mutation. Thus, the available data strongly support the view that expression of GFP-tubulin and its incorporation into microtubules does not detectably interfere with microtubule functions in yeast, and is therefore a valuable probe for analysis of microtubule behavior.

Heretofore, it has been extremely difficult to directly measure microtubule dynamics in mammalian cells throughout the cell cycle because of the difficulty of coordinating microinjection of fluorescent tubulin with the cell cycle and the fact that mitotic cells represent only a small fraction of the cells in a population. Other methods to visualize individual microtubules, such as differential interference contrast microscopy, are also more difficult in mitotic cells given their generally rounded morphology (Hayden et al., 1990). Cells expressing GFP-tubulin have the potential to be an invaluable tool for studying microtubule dynamics, organization, and behavior throughout the cell cycle. To date, transient expression of GFP-tagged mouse β6-tubulin in cultured cells strongly suggests that microtubule dynamic behavior is not altered by expression of the GFP construct, although quantitative analysis of microtubule dynamics in these cells was not performed (Ludin and Matus, 1998 Heidemann et al., 1999). In this work, we demonstrate that a cell line permanently expressing GFP-tubulin can be prepared and that the dynamic behavior of interphase microtubules in these cells is very similar to that in parental cells injected with rhodamine-labeled tubulin. We have used these cells to directly measure the changes in microtubule behavior throughout the cell cycle. In contrast to previous results inXenopus egg extracts (Belmont et al., 1990 Verdeet al., 1992 Tournebize et al., 2000), our results demonstrate that both the frequency of catastrophe and of rescue are altered in mitotic compared with interphase cells. The percentage of time microtubules spend in an attenuated state, or paused, is also dramatically reduced in mitotic cells. The rates of elongation and rapid shortening are not changed. In addition to quantification of microtubule dynamic instability in mitotic cells, we document microtubule release from the centrosome and microtubule tethering at the cell cortex. The availability of cells expressing GFP-tubulin should provide a simple, easily manipulated system to examine microtubule behavior in mammalian cells.


12.4: Microtubules - Biology

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Microtubules the thickest cytoskeletal elements in cells are hollow structures that consist of paired globular proteins, alpha and beta tubulins.

These heterodimers form linear rows called protofilaments, which have structural polarity. Meaning that each array is arranged with plus and minus ends. On the plus end where beta tubulins are exposed dimers are added. In contract, on the minus side where alpha tubulins are outward facing dissociation occurs.

However, in other cases microtubules secure stability by directly binding with different proteins like microtubule-associated proteins.

In addition their polarity allows for directional movement throughout the cytoplasm as is the case with dynein and kinesin motor proteins that efficiently transport various cargoes like vesicles.

Microtubules are also key components of cilia and flagella which are specialized extensions that move fluid over the surface of stationary cells and function as propellers in other cells moving them throughout their environments.

In the end, whether they're involved in chromosomal separation during cell division, transporting vesicles in the brain, or sweeping debris out of the lungs microtubules are essential for the growth and development, organizational strength and support, and motility that cells need.

4.10: Microtubules

There are three types of cytoskeletal structures in eukaryotic cells&mdashmicrofilaments, intermediate filaments, and microtubules. With a diameter of about 25 nm, microtubules are the thickest of these fibers. Microtubules carry out a variety of functions that include cell structure and support, transport of organelles, cell motility (movement), and the separation of chromosomes during cell division.

Microtubules are hollow tubes whose walls are made up of globular tubulin proteins. Each tubulin molecule is a heterodimer, consisting of a subunit of &alpha-tubulin and a subunit of &beta-tubulin. The dimers are arranged in linear rows called protofilaments. A microtubule usually consists of 13 protofilaments, arranged side by side, wrapped around the hollow core.

Because of this arrangement, microtubules are polar, meaning that they have different ends. The plus end has &beta-tubulin exposed, and the minus end has &alpha-tubulin exposed. Microtubules can rapidly assemble&mdashgrow in length through polymerization of tubulin molecules&mdashand disassemble. The two ends behave differently in this regard. The plus end is typically the fast-growing end or the end where tubulin is added, and the minus end is the slow-growing end or the end where tubulin dissociates&mdashdepending on the situation.

This process of dynamic instability, where microtubules rapidly grow and shrink, is important for functions such as the remodeling of the cytoskeleton during cell division and the extension of axons from growing neurons.

Microtubules also can be stable, often by binding to microtubule-associated proteins, which help the cell to maintain its shape. Other proteins, called motor proteins, can interact with microtubules to transport organelles in a particular direction. For example, many neurotransmitters are packaged into vesicles in the cell body of a neuron and are then transported down the axon along a &ldquotrack&rdquo of microtubules, delivering the vesicles to where they are needed. Finally, microtubules can also protrude outside of the cell&mdashmaking up the filamentous flagella and cilia that move to push cells (such as sperm) along, or to move fluid across their surfaces, such as in the lungs.

Brouhard, Gary J., and Luke M. Rice. &ldquoMicrotubule Dynamics: An Interplay of Biochemistry and Mechanics.&rdquo Ulasan Alam. Biologi Sel Molekul 19, tidak. 7 (July 2018): 451&ndash63. [Sumber]

Hashimoto, Takashi. &ldquoMicrotubules in Plants.&rdquo The Arabidopsis Book / American Society of Plant Biologists 13 (April 27, 2015). [Sumber]


Weekly Reflection (12/4-8)

This diagram is of the phospholipid bilayer and how different parts of the cell interact with each other.

During this week, we focused primarily on cell structure and how different parts of the cell work with each other. We also did an in class worksheet on cell parts and watched a video about the interior of a white blood cell (leukocyte). We lectured on cell structure as well which I found helpful.

The cell membrane is made up of a phospholipid bilayer and proteins and is an important part in keeping the shape of a cell. component in maintaining the shape of a cell. This part of cell helps to create a “boundary” which helps to monitor what is being let into and out of the cell through the bilayer. Only certain organic material that is able to pass through the semipermeable membrane can enter the cell. Excess or unwanted materials can be pushed out of the cell through the lipidbilayer if the cell has no use for it anymore. The (phospholipid)bilayer creates fluidity because of the constant movemenet of the phopholipids (heads and tails). Another key component of the membrane is cholesterol molecules. They are in charge of acting as a temperature buffer to maintain the fluidity of the cell. Both integral and peripheral proteins can be found on the membrane and are responsible for letting different materials in and out of the cell. The difference between intergal proteins penetrate the bilayer and while peripheral proteins on the other hand do not penetrate the bilayer. Both help with cell to cell recognition, transportation, signal transduction, and varies enzymatic activity. Another component to the cell membrane is signal transduction which is the receipt of chemical messages from the environment and the relay of those messages into the cell for response. An example of this is how animal cells rely on cell mebranes, but other cells have cell walls. The other type of cell, plant cells, have walls are made up of cellulose, fungal cell walls are made up of chitin, and bacterial cell walls are made up of peptidoglycan. That is the crucial difference between both animal and plant cells.

This diagram shows the phospholipid bilayer and where the integral and peripheral proteins lie in the bilayer.

Another part of the cell that we talked about this week was the cytoskeleton. The Cytoskeleton is a network of structural proteins that extends throughout the cytoplasm. It’s made up of microtubles, actin filaments, and intermediate filaments. Microtubules are in charge of moving organelles, with help of motor proteins (transport proteins), throughout the cell. A way to think about Mircortubules is like a cable car track that helps to move material to different parts of the cell. The centrosome is special because it is only found in animal cells. It is where microtubules originate from. The last part of the cytoskeleton are the microfilaments. They are responsible for the changes in a cells shape which important with interactions with other cells. The intermediate filaments help to prevent tension and ground the nucleus. All parts of the cytoskeleton help to make up the structure of each and every cell in your body and helps to maintain consistency.

This picture is of the cytoskeleton. It shows the filaments, mitochondria, and microtubules.

The last part of the cell that we learned about this week was extracellular matrix (ECM) which is absolutely critical to a cell. The ECM is a network of connective proteins and protoglycan molecules outside of the cell membrane that help with cell anchorage and cell communication. The intercellular junction is a type of protein that helps to connect cells to other cells. The other type of protein that is part of the ECM are open junctions. They allow hydrophilic molecules or ions to pass through from cell to cell. They also help to to “glue” the cells together (helps with cell shape) and creates a waterproof seal between the multiple cells.

This past week, we talked about the ideas that connect to Big idea 3 and Big idea 4. Big idea 3 focuses on cell communication and transmission f signals (think signal transduction). Big idea 4 is how special molecules differentiate from each other.


Dilution-induced disassembly of microtubules: Relation to dynamic instability and the GTP cap

Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15-fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubules of 60 μm/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Jurnal Biologi Sel 107:1437–1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP-tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.


12.4: Microtubules - Biology

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Tonton videonya: Cell Organelles 2 Cytoskeleton (Oktober 2022).